www.nature.com/scientificreports

OPEN Anti‑Acanthamoeba synergistic efect of chlorhexidine and Garcinia mangostana extract or α‑mangostin against Acanthamoeba triangularis trophozoite and cyst forms Suthinee Sangkanu1, Watcharapong Mitsuwan1,2, Wilawan Mahabusarakam3, Tajudeen O. Jimoh4,5, Polrat Wilairatana6*, Ana Paula Girol7, Ajoy K. Verma8, Maria de Lourdes Pereira9, Mohammed Rahmatullah10, Christophe Wiart11, Abolghasem Siyadatpanah12, Roghayeh Norouzi13, Polydor Ngoy Mutombo14,15 & Veeranoot Nissapatorn1*

Acanthamoeba spp. can cause amoebic keratitis (AK). Chlorhexidine is efective for AK treatment as monotherapy, but with a relative failure on drug bioavailability in the deep corneal stroma. The combination of chlorhexidine and propamidine isethionate is recommended in the current AK treatment. However, the efectiveness of treatment depends on the parasite and virulence strains. This study aims to determine the potential of Garcinia mangostana pericarp extract and α-mangostin against Acanthamoeba triangularis, as well as the combination with chlorhexidine in the treatment of Acanthamoeba infection. The minimal inhibitory concentrations (MICs) of the extract and α-mangostin were assessed in trophozoites with 0.25 and 0.5 mg/mL, for cysts with 4 and 1 mg/mL, respectively. The MIC of the extract and α-mangostin inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. The extract and α-mangostin combined with chlorhexidine demonstrated good synergism, resulting in a reduction of 1/4–1/16 of the MIC. The SEM results showed that Acanthamoeba cells treated with a single drug and its combination caused damage to the cell membrane and irregular cell shapes. A good combination displayed by the extract or α-mangostin and chlorhexidine, described for the frst time. Therefore, this approach is promising as an alternative method for the management of Acanthamoeba infection in the future.

1School of Allied Health Sciences, Southeast Asia Water Team (SEA Water Team), World Union for Herbal Drug Discovery (WUHeDD), and Research Excellence Center for Innovation and Health Products (RECIHP), Walailak University, Nakhon Si Thammarat, Thailand. 2Akkhraratchakumari Veterinary College and Research Center of Excellence in Innovation of Essential Oil, Walailak University, Nakhon Si Thammarat, Thailand. 3Division of Physical Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand. 4Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. 5Department of Biochemistry, Habib Medical School, Islamic University in Uganda, Kampala, Uganda. 6Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 7Department of Biology, Faculty of Sciences, São Paulo State University, São Paulo, Brazil. 8Department of Microbiology, National Institute of Tuberculosis & Respiratory Diseases (NITRD), New Delhi, India. 9Department of Medical Sciences, CICECO-Aveiro Institute of Materials &, University of Aveiro, Aveiro, Portugal. 10Department of Biotechnology & Genetic Engineering, University of Development Alternative Lalmatia, Dhaka, Bangladesh. 11School of Pharmacy, University of Nottingham Malaysia Campus, Selangor, Malaysia. 12Ferdows School of Paramedical and Health, Birjand University of Medical Sciences, Birjand, Iran. 13Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran. 14School of Public Health and Community Medicine, UNSW Medicine, UNSW, Sydney, NSW, Australia. 15Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Australia. *email: [email protected]; [email protected]

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 1 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 1. Primary sequence alignment of the DF3 region using CLUSTAL W. Te region shown a subset of the total DF3 region which demonstrates the highest variation. Asterisks denote similarities and dashes denote gaps within the nucleotide sequences.

Acanthamoeba is an opportunistic amoeba distributed in diverse natural habitats­ 1. Tis organism have two main forms: the trophozoite, an invasive stage; and cyst, a highly resistant stage in a very harsh conditions­ 2. Based on the size, shape and features of cysts, Acanthamoeba spp. have been divided into three groups (I, II, III). However, Acanthamoeba spp. has been classifed into 22 diferent genotypes (T1–T22 Genotype) based on molecular technique, which used 18S rRNA gene sequencing­ 3–5. Among them, T4 genotyge is the most isolated in clinical and environmental samples, followed by genotypes T3 and T5. In addition, the T4 genotype is the most virulent because it has a signifcant potential for binding to host cells than other genotypes­ 6. Acanthamoeba spp. are the causative agents of amoebic keratitis (AK) and granulomatous amoebic encephali- tis (GAE). AK can cause permanent loss of vision­ 7. Te rate of infectious keratitis is becoming alarming in recent times, a problem that may be related to a sudden increase in the population of contact lens wearers­ 2 Similarly, Acanthamoeba encysts penetrated deeply into the corneal stroma­ 8 as such, the cyst wall becomes impervious to existing drugs, and this becomes a drawback for further studies in the areas of drug formulations and designed for this organism. Garcinia mangostana Linn. is generally known as mangosteen and belongs to the family Clusiaceae. It is a tropical tree, widely distributed in Southeast Asia­ 9. Te pericarps of this fruit are commonly used in traditional medicine to treat several diseases which are non-toxic and safe to ­use10,11. Major compound in the mango- steen pericarps is xanthone group, particularly α-mangostin, which exhibited antibacterial activity­ 12, antifungal activity­ 13, antioxidant activity­ 14 anti-cancer activity­ 15–17, anti-infammatory activity­ 18,19, antiparasitic ­activity20–22. To the best of our knowledge, we discover that there is no single report on the anti-Acanthamoeba activity of G. mangostana extract to date. Hence, our study sought to investigate the efective concentration of the G. mangostana extract and α-mangostin on the growth inhibition of Acanthamoeba spp. and to demonstrate its synergistic efects combined with chlorhexidine on anti-Acanthamoeba activity. Results Genotypic and species identifcation of Acanthamoeba isolate WU19001. Te partial nucleotide sequences of DF3 region of Acanthamoeba sp. WU19001 from our previous study aligned using CLUSTAL W and revealed the highest variation, as shown in Fig. 1. Te 18S rDNA sequences were subjected to phylogenetic analysis and species identifcation. It showed very similar patterns to Acanthamoeba triangularis KX232518.1 (99.74% similarity). Te sequence homology search for the 35 Acanthamoeba spp. in the National Center for Biotechnology Information (NCBI) database showed WU19001 formed Acanthamoeba genotype T4 cluster (Fig. 2). Te nucleotide sequence of WU19001 has been deposited at Genbank under the accession number MW647650.

Minimum inhibitory concentration (MIC). G. mangostana extract and α-mangostin were determined for their anti-Acanthamoeba potential by MIC using the microtiter dilution broth method. As shown in Table 1, the MICs of G. mangostana extract against A. triangularis trophozoites and cysts were 0.25 and 4 mg/mL. Te pure compound, α-mangostin, exhibited MIC values at 0.5 and 1 mg/mL for trophozoites and cysts. Te MIC values of chlorhexidine against trophozoites and cysts were 0.008 and 0.064 mg/mL, respectively.

Growth assay. Susceptibilities of A. triangularis to G. mangostana extract and α-mangostin were deter- mined by growth assay. A. triangularis was found to be signifcantly (p < 0.05) susceptible to the extract and α-mangostin at all concentrations when compared to control. Te extract and α-mangostin showed higher sus- ceptibility against Acanthamoeba trophozoites than cysts. (Fig. 3). Afer 72 h incubation, MIC values of the extract and α-mangostin could decrease the viability to 0.15 × ­105 and 0.1 × ­105 cells/mL of trophozoites as com- pared to the control (6.0 × ­105) (Fig. 3a,b). Afer 24 h of incubation, the number of Acanthamoeba cysts decreased to 2.3 × ­105 and 0.8 × ­105 cells/mL when treated with MIC concentration of the extract and α-mangostin, respectively. We observed the re-growth which reached to the number of viable cells of 1.4 × ­105 and 2.2 × ­105 cells/mL in the presence of the extract and α-mangostin at 72 h of incubation. However, the treated cysts showed a decrease in viable cells when compared with the control that reached up to 5.8 × ­105 cells/mL at 72 h of incubation (Fig. 3c,d).

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 2 Vol:.(1234567890) www.nature.com/scientificreports/

Figure 2. A phylogenetic tree was constructed using the neighbour-joining method with the Kimura two- parameter algorithm with bootstrapping values for 1000 replicates. Te tree was reconstructed by considering 35 Acanthamoeba spp. isolates with the reference strains from NCBI.

MIC (mg/mL) Antimicrobial agents Trophozoites Cysts G. mangostana extract 0.25 4 α-mangostin 0.5 1 Chlorhexidine 0.008 0.064

Table 1. Minimal inhibitory concentration (MIC) of G. mangostana extract, α-mangostin and chlorhexidine against A. triangularis trophozoites and cysts.

Synergistic efects. Te evaluation of the synergistic efects between the plant extract and the drug was determined in this study. For A. triangularis trophozoites, an additive interaction (FICI = 1) was observed in the combination of chlorhexidine and the extract. Chlorhexidine at 0.002 mg/mL was synergistic when combined with α-mangostin at 0.032, 0.062 and 0.125 mg/mL (Table 2). For the cystic form, chlorhexidine was synergistic when combined with the extract and α-mangostin. With the extract, synergism was observed in 0.004, 0.008 and 0.016 mg/mL of chlorhexidine when combined with the extract 0.5 and 1 mg/mL. In addition, chlorhexidine at 0.004 and 0.008 mg/mL was found to be synergistic with various concentrations (0.062, 0.125 and 0.25 mg/mL) of α-mangostin (Table 3). Te percentage of viability in A. triangularis in additive and synergistic efects was less than 10% (Figs. 4 and 5).

SEM analysis. Te alterations due to the action of G. mangostana extract and α-mangostin were confrmed by SEM micrograph are shown in Figs. 6 and 7. Control trophozoites (Fig. 6a) treated with 1% DMSO exhib- ited normal cells with acanthopodia. During the treatment with the extract (Fig. 6b), the damaged cells were

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 3 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 3. G. mangostana extract and α-mangostin inhibit the growth of A. triangularis trophozoites and cysts in vitro. To determine the efect of G. mangostana extract and α-mangostin on trophozoites (a,b) and on cysts (c,d), assays were performed by inoculating 2 × ­105 cells/mL in PYG medium in the presence of G. mangostana extract or α-mangostin at MIC, 0.5 × MIC and 0.25 × MIC fnal concentrations. Te inhibitory activity was carried out using trypan blue exclusion assay up to 72 h; 1% DMSO was used as a negative control.

MIC in combination (mg/mL) Combination agents Chlorhexidine Extract or α-mangostin FICI Efect G. mangostana extract 0.004 0.125 1 Additivity 0.002 0.032 0.31 Synergistic α-mangostin 0.002 0.062 0.37 Synergistic 0.002 0.125 0.50 Synergistic

Table 2. Fractional inhibitory concentration index (FICI) of the combination test in targeting A. triangularis trophozoites.

observed as fat and smooth surface. For treatment with MIC of chlorhexidine and α-mangostin, the trophozo- ites reduced in size, shrunken appearance, loss of acanthapodia, and having pores-like structure on the surface as shown in Fig. 6c,d. Trophozoites treated with the combination of chlorhexidine and the extract at FICI equal 1, SEM showed rounded morphology with pores on the cell surface (Fig. 6e). In combination with chlorhex- idine and α-mangostin, trophozoites have died completely with a rough surface and tiny when compared to the control (Fig. 6f). Also, micrographs of control cysts were intact with oval shape and smooth surface as shown in Fig. 7a. Te oval cysts of A. triangularis were fat, irregular in shape, causing a collapse of the ectocyst walls afer treatment with chlorhexidine, the extract, α-mangostin and in combination (Fig. 7b–f).

Toxicity. At low concentrations, ranging from 0.032–0.062 mg/mL for G. mangostana extract, the number of living cells were constant at 24 h. However, the survival of Vero cells tended to be lower than those afer treat- ments with the extract (0.125–4 mg/mL) (Fig. S2a). For α-mangostin, the surviving Vero cells were observed at 0.008–0.016 mg/mL (non-toxic) but decreased at the concentrations of 0.032–1 mg/mL (Fig. S2b). Te efect of α-mangostin on Vero cells was further determined according to ­IC50 value, which was obtained from the viable interpolation of the cell line. Pure α-mangostin exhibited an inhibitory efect on Vero cells with IC­ 50 value 0.016 mg/mL (39 μM) afer 24 h of treatment (Fig. S2c). To reduce the toxicity, a combination sets of α-mangostin and chlorhexidine treatment was further challenged to determine the survival of Vero cells. How- ever, it was clearly observed the decreased survial when the concentration of α-mangostin was higher than 0.016 mg/mL (data were not shown).

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 4 Vol:.(1234567890) www.nature.com/scientificreports/

MIC in combination (mg/mL) Combination agents Chlorhexidine Extract or α-mangostin FICIFICI Efect 0.004 0.5 0.18 Synergistic 0.004 1 0.31 Synergistic G. mangostana extract 0.008 0.5 0.24 Synergistic 0.008 1 0.37 Synergistic 0.016 0.5 0.36 Synergistic 0.004 0.062 0.12 Synergistic 0.004 0.125 0.18 Synergistic 0.004 0.250 0.31 Synergistic 0.008 0.062 0.18 Synergistic α-mangostin 0.008 0.125 0.24 Synergistic 0.008 0.250 0.37 Synergistic 0.008 0.125 0.24 Synergistic 0.008 0.062 0.18 Synergistic

Table 3. Fractional inhibitory concentration index (FICI) of the combination test in targeting A. triangularis cysts.

Figure 4. Combination of G. mangostana extract or α-mangostin with chlorhexidine for anti-amoebic efects on trophozoites of A. triangularis. Parasites were grown in PYG medium in the presence of chlorhexidine alone and combination with G. mangostana extract (a) or α-mangostin (b) for 24 h. Te inhibitory activity was carried out using trypan blue exclusion assay; 1% DMSO was used as negative control (c). Treated trophozoites were observed under inverted microscopy (20x). Te relative percentage of viability was defned as (mean of the treated /mean of the control) × 100. *p < 0.05, statistically signifcant diference in combination to single drug treatment.

Discussion Te herb-drug interaction may impact to the potential health-promoting efect of increasing drug efcacy or decreasing common adverse efect. As such, we present in this study an efective treatment for A. triangularis infection with herb-drug based combination strategy. Also, we highlight the potential of the anti-Acanthamoeba activity of G. mangostana which also known as the “queen of fruits”. In previous literature, xanthones is a major phytochemical compound from G. mangostana which have been presented in several parts of plant such as pericarp, whole fruits, leaves and ­bark6. Te pericarp of mangosteen

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 5 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 5. Combination of G. mangostana extract or α-mangostin with chlorhexidine for anti-amoebic efects on cysts of A. triangularis. Parasites were grown in PYG medium in the presence of chlorhexidine alone and combination with G. mangostana extract (a) or α-mangostin (b) for 24 h. Te inhibitory activity was carried out using trypan blue exclusion assay; 1% DMSO was used as a negative control (c). Treated cysts were observed under inverted microscopy (40x). Te relative percentage of viability was defned as (mean of the treated /mean of the control) × 100. *p < 0.05, statistically signifcant diference in combination to single drug treatment.

fruit are the most abundant of xanthones α-mangostin (78% total xanthone content)23. In addition, there are various components of other xanthones, such as β-mangostin, gartanin, 8-deoxygartanin, garcinones A, B, C, D and E, mangostinone, 9-hydroxycalabaxanthone, and isomangostin­ 6,24. Nevertheless, previous studies have reported that the concentration of α-mangostin correlates with the biological activity of mangosteen ­extract10. Interestingly, the compound α-mangostin from G. mangostana has ever been reported its property against other parasites like Plasmodium falciparum22. Some studies have reported the efects of G. mangostana extract and α-mangostin on anti-parasitic ­activity22,25, but the efect on Acanthamoeba spp. has so far never been explored. Our study therefore demonstrated for the frst time of G. mangostana extract from the pericarp and α-mangostin exhibit anti-Acanthamoeba activity against A. triangularis trophozoites and cysts. A. triangularis WU19001 was a selected strain parasite used in this study due to its characteristic property belong to group II (genotype T4). Group 2 Acanthamoeba species are pathogenic in nature, typically having double-walled with a wrinkled ectocyst and stellate polygonal, triangular, or oval endocyst­ 26. A. triangularis was originally isolated from human faeces­ 27 and currently discovered from environmental water souces, as reported in this study. In 2008, the pathogenic strain of A. triangularis has frstly reported in a clinically confrmed case of Acanthamoeba keratitis in ­Korea28. Due to climate change and global warming, it is postulated that A. triangularis would be more likely to be associated to clinical scenario in times to come as it is widely contaminated in the environment. In fact, eradicating Acanthamoeba infection seems impossible due to the high resistance of the cysts to anti-Acanthamoeba drugs. Terefore, we also investigated the efective concentration of G. mangostana extract and α-mangostin against A. triangualris trophozoite and cyst stages. Our results showed that the extract and α-mangostin can inhibit A. triangularis in PYG medium. It is noteworthy that the number of Acanthamoeba trophozoites and cysts were signifcantly reduced (p < 0.05) in the treatment with MIC concentrations of the extract and α-mangostin. Tey were found to have the greatest growth-inhibitor for trophozoites at 72 h. For the cystic form, they survived on 72 h (Fig. 3) which might be due to the decreased the decreased efect of the extract and α-mangostin. In addition, the cyst form is a is a dormant stage against severe condition, including the presence of anti-Acanthamoeba agents­ 29. Overall, this study therefore suggests that the extract and α-mangostin are promising agents that shows the remarkable efects against A. triangularis infections. However, the develop- ment method for support the stability of the extract and α-mangostin has been validated since it is important for further pharmacokinetic or tissue distribution ex vivo studies. Chlorhexidine is the drug of choice for treatment Acanthamoeba keratitis, since it is efective against both trophozoites and cysts­ 8. In this study, a single drug chlorhexidine was studied on anti-Acanthamoeba activity

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 6 Vol:.(1234567890) www.nature.com/scientificreports/

Figure 6. Scanning micrographs of A. triangularis trophozoites afer treatment with G. mangostana extract, chlorhexidine, α-mangostin and in combination at 24 h. Control trophozoites (a), trophozoites treated with G. mangostana extract (1 mg/mL) (b), α-mangostin (0.5 mg/mL) (c), chlorhexidine (0.008 mg/mL) (d), and combination of chlorhexidine (0.004 mg/mL) and G. mangostana extract (0.125 mg/mL) (e), combination of chlorhexidine (0.002 mg/mL) and α-mangostin (0.032 mg/mL) (f). Magnifcation: (a–f) = × 5000.

and prosperously exhibited inhibitory activity in A. triangularis trophozoites and cysts with MIC values of 0.008 and 0.064 mg/mL (Table 1), respectively. According to an earlier report, chlorhexidine showed amoebicidal and cysticidal properties at 200 μg/mL (0.02%), but it exhibited side ­efects30. In fact, a single drug, used to treat infec- tious diseases, includes this parasitic infection that causes side efects, long-term for clearance use, high cost and drug-resistant parasites. Terefore, the combination approach is constantly being introduced to fnd these pitfalls. Chlorhexidine has ofen been used in combination with aromatic diamidines­ 8, aminoglycosides, imidazoles, and polyene­ 31. However, these chemicals have side efects on keratocytes found in cases of human keratitis­ 32. Recently, the discovery of compounds with anti-Acanthamoeba activity in plants and herbs has been very encouraging to evaluate a source of secondary metabolites with anti-Acanthamoeba efects. Te combination test for possible synergistic efects against Acanthamoeba spp. was considered to reduce the MIC of the drug. To support this, our study also revealed the efect of the combination of G. mangostana extract and chlorhexidine against A. triangu- laris trophozoites and cysts. Chlorhexidine showed an additive efect when combined with the G. mangostana extract. An additive efect occurs when the substance added to increase or improve the efectiveness but not to the extent of synergistic ­interaction33. G. mangostana extract was combined with chlorhexidine to produce a synergistic efect against A. triangularis cysts. Te FICI values demonstrated the synergy for concentration of 0.004 to 0.016 mg/mL of chlorhexidine and 0.5 to 1 mg/mL of G. mangostana extract as shown the viability of less than 10%. Te α-mangostin was found to be more efective when combined with chlorhexidine. It is inter- esting to note that the concentration of chlorhexidine can be reduce by 1/4–1/16 of the MIC in the presence of α-mangostin. Nowadays, it is difcult and expensive to develop new drugs, therefore it has been considered the fnding of alternative strategies to reduce toxicity and/or the development of resistance pathogens. From this study, it appears to be a promising combined chlorhexidine with G. mangostana extract or α-mangostin to fght infection and especially the resistance pattern of Acanthamoeba spp. in the future. Te mode of action considered in this study was confrmed by scanning electron microscopy (SEM), as shown in Figs. 6 and 7. Treated trophozoites showed similar fat cells and smooth surfaces as a result of the total destruc- tion of acanthopodia in the presence of extract and chlorhexidine. Regarding the combination of the drug and G. mangostana extract or α-mangostin, the morphology of trophozoites being observed in the presence of pores on their surface and cells were rounded and small. Chlorhexidine is positively and ionically charged with the parasite´s negatively charged plasma membrane, resulting in the membrane structure that gives rise to perme- ability modulation, ionic leakage and cytoplasmic disruptions causing cellular damage and cell death­ 34,35. Control cysts showed regular morphological characteristics. Overall, A. triangularis cysts were fat and morphologically

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 7 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 7. Scanning micrographs of A. triangularis cysts afer treatment with G. mangostana extract, chlorhexidine, α-mangostin and in combination at 24 h. Control cysts (a), cysts were treated with G. mangostana extract (16 mg/mL) (b), α-mangostin (4 mg/mL) (c), chlorhexidine (2.56 mg/mL) (d), and combination of chlorhexidine (0.004 mg/mL) and G. mangostana extract (0.5 mg/mL) (e), combination of chlorhexidine (0.04 mg/mL) and α-mangostin (0.062 mg/mL) (f). Magnifcation: (a–f) = × 5000.

deformed (of irregular shape and size) as a result of the destruction of the ectocyst walls afer the treatment given with G. mangostana extract, α-mangostin, chlorhexidine and in combination. For a pure compound, α-mangostin exhibited toxicity to Vero cells at ­IC50 values 39 μM (0.016 mg/mL). Our preliminary fnding is however surprisingly diferent from a previous study reported that α-mangostin was non-toxic to nontumorigenic human pancreatic duct epithelial cells, at a dose as high as 40 μM36. In addition, xanthones in G. mangostana pericarp proved to be non-toxic to mice in vivo when administered orally at a dose of 100 mg/kg of body weight/day for 7 ­days37. Since α-mangostin is gaining more popular to be used against infectious diseases, therefore, it strongly suggests for further comprehensive studies such as chemical structure modifcation and structure–activity relationship as well as nanotechnology to evaluate the mechanism of action of these compounds against Acanthamoeba spp., before any conclusion could be made. Besed on the results obtained from this study, ethanolic extract of G. mangostana from the pericarp and α-mangostin possess anti-Acanthamoeba activities against A. triangularis trophozoite and cyst stages. Moreover, the present study focuses on the novel combination for treatment of Acanthamoeba infection. Te combination of G. mangostana extract and α-mangostin with chlorhexidine generated synergistic efects which increased efectively for the treatment of Acantrhamoeba infection. Materials and methods Preparation of plant extracts. Te 50 g of dry G. mangostana pericarp powder was soaked in 200 mL of ethanol for 7 days. Te extract was fltered through Whatman No. 1 (GE Healthcare Life Science, Bucking- hamshire HP7 9NA, United Kingdom) using a vacuum and pressure pump. Te solution was evaporated to dry- ness under reduced pressure using a rotary evaporator to obtain G. mangostana extract. Te 200 mg of extract was subjected to column chromatography using silica gel as a stationary phase and eluted with 20% acetone in 750 mL of hexane. Te collected fractions were chromatographed on a silica gel thin-layer chromatography compared with the authentic compound, α-mangostin. Fractions with one spot of α-mangostin on chroma- togram were combined. Te solvent was removed to give 30 mg yellow solid of α-mangostin. Te extract and α-mangostin were dissolved in 99.5% DMSO and stored at − 20 °C until use.

Cultivation of A. triangularis. A. triangularis WU19001, a strain from the recreational reservoir at Walailak University, Nakhon Si Tammarat-Tailand, was used in this ­study38. Te parasite was grown in PYG medium [20 g proteose peptone, 2 g yeast extract, 0.98 g MgSO­ 4·7H2O, 0.35 g Na­ 2HPO4·7H2O, 0.34 g KH­ 2PO4, 0.02 g ­(NH4)2Fe(SO4)2·6H2O, 18 g glucose]. Te trophozoites were observed afer 72 h of incubation at room

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 8 Vol:.(1234567890) www.nature.com/scientificreports/

temperature and were cultured in this medium for 1 week Te 90% mature cysts were obtained PYG medium. Te cysts were harvested when the cultures were incubated for at least 1 week without addition fresh medium. Te parasite reproduced exponentially until they reached the maximum level of 1 × ­106 cells/mL, afer which a reduced nutrients led to encystation (cyst formation) due to unfavorable conditions for the parasite’s growth especially into trophozoite stage. At the end, all fully homogenic inoculum of mature cysts were successfully har- vested. Trophozoites and cysts were centrifuged at 4000 rpm for 5 min and re-suspended in fresh PYG thereafer. For counting, 50 μL of cell suspension was mixed with 50 μL of trypan blue. Viability was investigated under the inverted microscope based on the principle of the dye can cross the membrane of dead cells with blue color, but not intact membrane of viable cells with colorless appearances.

Minimal inhibitory concentration (MIC). Te minimum inhibitory concentration (MIC) for G. man- gostana extract and α-mangostin was determined using the microtiter broth dilution ­method38. Te extract and α-mangostin were diluted to give a fnal concentration of 4, 2, 1, 0.5, 0.25, 0.125, 0.062 mg/mL in a 96-well microplate. Ten 100 µL of 2 × ­105 cells/mL of trophozoites and cysts were inoculated into each well. Chlorhex- idine and 1% DMSO were included as a positive and negative control, respectively. Te plates were incubated at room temperature for 24 h. Te viability of parasites was calculated as follows: % viability = (mean of the viable parasite/control) × 100. Te MIC value was defned as the lowest concentration that inhibited > 90% of viable growth when compared with the control.

Growth assay. In the present study, the growth inhibition on A. triangularis of G. mangostana extract and α-mangostin was carried out following the procedure that previously described­ 1 with modifcations. Te tropho- zoites and cysts (2 × ­105 cells/mL) were incubated with the extract and α-mangostin in the MIC, 0.5 × MIC and 0.25 × MIC, except for untreated control tubes, which had only PYG medium and incubated at room tempera- ture for 72 h. At 24 h intervals, the viability of parasite was determined by staining with 0.2% trypan blue.

Drug combinations. Te checkerboard ­method39 was used to evaluate the interaction between G. man- gostana extract/α-mangostin and chlorhexidine against A. triangularis. Subsequently, the microdilution assay was performed in a 96-well plate with a fnal volume of 200 µL. Te extract, α-mangostin and chlorhexidine were diluted with PYG to obtain 4 times to their fnal concentrations of 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC and MIC. A total of 100 µL of the extract + chlorhexidine or α-mangostin + chlorhexidine were prepared in 96-well plate and added 100 µL of parasite suspension containing 2 × ­105 cells/mL into the wells. Te plates were incubated at room temperature for 24 h. Te viability of parasite was defned as the lowest concentration that inhibited > 90% of growth when compared to the negative control. Te assessment of the results was defned as the Fractional Inhibitory Concentration Index (FICI), which was calculated using the following: FICI of combination = FIC A + FIC B

FIC A = MIC of chlorhexidine in combination/MIC of chlorhexidine alone

FIC B = MIC of extract in combination/MIC of extract alone.

The combination was considered synergistic for FICI ≤ 0.5, additive for 0.5 < FICI ≤ 1, indifferent for 1 < FICI < 4, and antagonistic for FICI ≥ 4, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) defnition.

Scanning electron microscopic (SEM) study. Trophozoites and cysts of A. triangularis were treated with chlorhexidine, G. mangostana extract, α-mangostin and in combinations. Afer incubation, cells were col- lected by centrifugation at 4000 rpm for 5 min and re-suspended in phosphate bufer saline (PBS). Cells in 1% DMSO were used as negative controls. Samples were fxed with 2.5% glutaraldehyde overnight. Te samples were further dehydrated with a series of graded alcohol (20%, 40%, 60%, 80%, 90%, and 100% ethanol), mounted on aluminum stubs, and allowed to dry using a critical point dryer. Samples were then coated with gold particles and the morphology of A. triangularis trophozoites and cysts afer treatment was subsequently examined under SEM (SEM-Zeiss, Munich, Germany) at the Center for Scientifc and Technological Equipment, Walailak Uni- versity, Nakhon Si Tammarat, ­Tailand38.

Toxicity. Te cytotoxic efects of G. mangostana extract, α-mangostin, chlorhexidine and combination sets were evaluated using the Vero cell line (8200F270602). Cells were cultured in Dulbecco’s Modifed Eagle’s (DMEM) medium (Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS and 1% antibiotic con- taining penicillin G of 100 units/mL, streptomycin of 100 μg/mL. Te culture was incubated at 37 °C in a humid- ifed atmosphere and 5% ­CO2. Afer the cells reached 90% confuence, detachment was performed using trypsin 4 ethylene diamine tetraacetic acid (EDTA), incubated at 37 °C in 5% CO­ 2. Single cells at a density of 1.5 × ­10 cells/100 μL were seeded into each well of a 96-well polystyrene plate and allowed to attach for 24 h. Ten, 100 μL of the extract and α-mangostin, previously prepared at multiple concentrations, chlorhexidine and combination sets were gently added. Afer incubation for 24 h, the cytotoxic efects were determined using the MTT assay. Te absorbance was measured using a microplate reader (Biotek, Cork, Ireland) at 570 nm. Te percent survival was calculated using the following equation:

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 9 Vol.:(0123456789) www.nature.com/scientificreports/

% survival = (ABt/ABu) × 100

ABt and ABu denote the absorbance values of treated and untreated cells, ­respectively40.

Statistical analysis. Te experiments were performed in triplicate. All data were recorded and entered using the statistical package sofware (SPSS Inc. Chicago, IL, USA). Te data were expressed as mean ± SD. Sta- tistical analysis was analyzed by the two-tailed unpaired Student’s t-test. In all analyzes, p < 0.05 was considered statistically signifcant.

Received: 5 December 2020; Accepted: 25 March 2021

References 1. Chu, D., Miles, H., Toney, D., Ngyuen, C. & Marciano-Cabral, F. Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp. Parasitol. Res. 84, 746–752 (1998). 2. Sifaoui, I. et al. Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure. Exp. Parasitol. 183, 117–123 (2017). 3. Tice, A. K. et al. Expansion of the molecular and morphological diversity of Acanthamoebidae (Centramoebida, Amoebozoa) and identifcation of a novel life cycle type within the group. Biol. Direct. 11, 69. https://​doi.​org/​10.​1186/​s13062-​016-​0171-0 (2016). 4. Haniloo, A., Pezeshki, A., Mahmmodzadeh, A. & Kadkhodamohammadi, E. Genotyping of Acanthamoeba spp. from water sources from Northwestern Iran. Acta Parasitol. 62, 790–795 (2017). 5. Coronado-Velázquez, D. et al. Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior. Parasitol. Int. 74, 102002. https://​doi.​org/​10.​1016/j.​parint.​2019.​102002 (2019). 6. Chao, M., Tongseesuksai, T., Boonmars, T. & Laummaunwai, P. Investigation of the in vitro cysticidal activity of miltefosine against Acanthamoeba spp. J. Parasites Dis. 44, 491–495 (2020). 7. Walochnik, J. et al. Granulomatous amoebic encephalitis caused by Acanthamoeba amoebae of genotype T2 in a human immu- nodefciency virus-negative patient. J. Clin. Microbiol. 46, 338–340 (2008). 8. Lorenzo-Morales, J., Khan, N. A. & Walochnik, J. An update on Acanthamoeba keratitis: Diagnosis, pathogenesis and treatment. Parasites 22, 10. https://​doi.​org/​10.​1051/​paras​ite/​20150​10 (2015). 9. Taher, M., Tg Zakaria, T., Susanti, D. & Zakaria, Z. A. Hypoglycaemic activity of ethanolic extract of Garcinia mangostana Linn. in normoglycaemic and streptozotocin-induced diabetic rats. BMC Complement. Altern. Med. 16, 135. https://​doi.​org/​10.​1186/​ s12906-​016-​1118-9 (2016). 10. Ibrahim, M. Y., Hashim, N. M. & Mariod, A. A. A. α-mangostin from Garcinia mangostana Linn: An updated review of its phar- macological properties. Arab. J. Chem. 9, 317–329 (2016). 11. Sunarjo, L., Suharti, O. & Susanto, H. S. Te preliminary study on safety of using mangosteen peel extract as natural herbs. JMSCR 50, 24851–24856 (2017). 12. Sakagami, Y., Iinuma, M., Piyasena, K. G. & Dharmaratne, H. R. Antibacterial activity of alpha-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics. Phytomedicine 12, 203–208 (2005). 13. Puripattanavong, J. K. W., Khajorndetkun, W. & Chansathirapanich, W. Improved isolation of α-mangostin from the fruit hull of Garcinia mangostana and its antioxidant and antifungal activity. Planta Med. 72, 1078. https://​doi.​org/​10.​1055/S-​2006-​950128 (2006). 14. Husen, S. A., Khaleyla, F., Ansori, A. N. M., Susilo, R. J. K. & Winarni, D. Antioxidant activity assay of alpha-mangostin for ame- lioration of kidney structure and function in diabetic mice. ASSEHR 98, 84–88 (2018). 15. Aisha, A. F., Abu-Salah, K. M., Ismail, Z. & Majid, A. M. In vitro and in vivo anti-colon cancer efects of Garcinia mangostana xanthones extract. BMC Complement. Altern. Med. 12, 104–112 (2012). 16. Wang, J. J., Sanderson, B. J. & Zhang, W. Cytotoxic efect of xanthones from pericarp of the tropical fruit mangosteen (Garcinia mangostana Linn.) on human melanoma cells. Food Chem. Toxicol. 49, 2385–2391 (2011). 17. Matsumoto, K. et al. Induction of apoptosis by xanthones from mangosteen in human leukemia cell lines. J. Nat. Prod. 66, 1124–1127 (2003). 18. Chen, L. G., Yang, L. L. & Wang, C. C. Anti-infammatory activity of mangostins from Garcinia mangostana. Food Chem. Toxicol. 46, 688–693 (2008). 19. Fu, Y., Zhou, H., Wang, M., Cen, J. & Wei, Q. Immune regulation and anti-infammatory efects of isogarcinol extracted from Garcinia mangostana L. against collagen-induced arthritis. J. Agric. Food Chem. 62, 4127–4134 (2014). 20. Riscoe, M., Kelly, J. X. & Winter, R. Xanthones as antimalarial agents: Discovery, mode of action, and optimization. Curr. Med. Chem. 12, 2539–2549 (2005). 21. Azebaze, A. G. et al. Prenylated xanthone derivatives with antiplasmodial activity from Allanblackia monticola STANER L.C. Chem. Pharm. Bull. 54, 111–113 (2006). 22. Mahabusarakam, W., Kuaha, K., Wilairat, P. & Taylor, W. C. Prenylated xanthones as potential antiplasmodial substances. Planta Med. 72, 912–916 (2006). 23. Ansori, A. N. M. et al. A review on medicinal properties of mangosteen (Garcinia mangostana L.). Res. J. Pharm. Technol. 13, 974–982 (2020). 24. Gutierrez-Orozco, F. & Failla, M. L. Biological activities and bioavailability of mangosteen xanthones: A critical review of the current evidence. Nutrients 5, 3163–3183 (2013). 25. Upegui, Y. et al. In vivo antimalarial activity of α-mangostin and the new xanthone δ-mangostin. Phytother Res. 29, 1195–1201 (2015). 26. Marciano-Cabral, F. & Cabral, G. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16, 273–307 (2003). 27. Pussard, M. & Pons, R. Morphologie de la paroi kystique et taxonomie du genre Acanthamoeba (Protozoa, Amoebida). Protistologica 13, 557–598 (1977). 28. Xuan, Y. H. et al. Keratitis by Acanthamoeba triangularis: Report of cases and characterization of isolates. Korean J. Parasitol. 46, 157–164 (2008). 29. Bunsuwansakul, C. et al. Acanthamoeba in Southeast Asia: Overview and Challenges. Korean J. Parasitol. 57, 341–357 (2019). 30. Siddiqui, R., Aqeel, Y. & Khan, N. A. Te development of drugs against Acanthamoeba infections. Antimicrob. Agents Chemother. 60, 6441–6450 (2016). 31. Fakae, L. B., Stevenson, C. W., Zhu, X. Q. & Elsheikha, H. M. In vitro activity of Camellia sinensis (green tea) against trophozoites and cysts of Acanthamoeba castellanii. Int. J. Parasitol. Drugs Drug Resist. 13, 59–72 (2020).

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 10 Vol:.(1234567890) www.nature.com/scientificreports/

32. Anwar, A. et al. Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and efects of conjugation with silver nanoparticles. Parasitol. Res. 119, 1943–1954 (2020). 33. Cheesman, M. J., Ilanko, A., Blonk, B. & Cock, I. E. Developing new antimicrobial therapies: Are synergistic combinations of plant extracts/compounds with conventional antibiotics the solution?. Pharmacogn. Rev. 11, 57–72 (2017). 34. Elsheikha, H. M., Siddiqui, R. & Khan, N. A. Drug discovery against Acanthamoeba infections: Present knowledge and unmet needs. Pathogens 9, 405. https://​doi.​org/​10.​3390/​patho​gens9​050405 (2020). 35. Fatimah, H. & Nakisah, M. A. Visualization on the efect of chlorhexidine gluconate, a biocide on Acanthamoeba sp. by electron microscopy. Malays. J. Microsc. 9, 154–159 (2013). 36. Hafeez, B. B. et al. α-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograf mouse model. Antioxid. Redox Signal. 21, 682–699 (2014). 37. Kaomongkolgit, R., Jamdee, K., Pumklin, J. & Pavasant, P. Laboratory evaluation of the antibacterial and cytotoxic efect of alpha- mangostin when used as a root canal irrigant. Indian J Dent. 4, 12–17 (2013). 38. Mitsuwan, W. et al. Curcuma longa ethanol extract and Curcumin inhibit the growth of Acanthamoeba triangularis trophozoites and cysts isolated from water reservoirs at Walailak University,Tailand. Pathog Glob. Health. 114, 194–204 (2020). 39. Lorian, V. Antibiotics. In Laboratory Medicine (ed. Lorian, V.) 69–71 (Springer, Baltimore, 1996). 40. Kaomongkolgit, R., Jamdee, K. & Chaisomboon, N. Antifungal activity of alpha-mangostin against Candida albicans. J. Oral Sci. 51, 401–406 (2009). Acknowledgements We highly appreciate for the support of Te Royal Patronage of Her Royal Highness Princess Maha Chakri Sirind- horn—Botanical Garden of Walailak University, Nakhon Si Tammarat under the project entitled: Medicinal under-exploited Tai native plant against Acanthamoeba, Leishmania donovani, and Plasmodium falciparum— Toward South East Asia collaboration initiative (WUBG020-2564), Tailand, Research Institute of Health Sci- ence (RIHS), Walailak University for the laboratory facilities, and Assoc. Prof. Dr. Chuchard Punsawad, School of Medicine, Walailak University for providing human Vero cell line. We would also like to acknowledge the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 and UIDP/50011/2020, national funds by FCT/MCTES. Author contributions S.S. performed all the laboratory work, analyzed the data and wrote a draf of the paper; P.W. and V.N. conceived the idea, designed the experiments related to natural products and parasitology, and overlooked in the labora- tory work; W.M. assisted in the experiments; All authors read, reviewed, edited, agreed, and approved for the submission of the fnalized manuscript.

Competing interests Te authors declare no competing interests. Additional information Supplementary Information Te online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​87381-x. Correspondence and requests for materials should be addressed to P.W. or V.N. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

© Te Author(s) 2021

Scientifc Reports | (2021) 11:8053 | https://doi.org/10.1038/s41598-021-87381-x 11 Vol.:(0123456789)